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| Home > Publications database > Genetic control of carbohydrate uptake and utilization in Corynebacterium glutamicum |
| Home > Publications database > Genetic control of carbohydrate uptake and utilization in Corynebacterium glutamicum |
| Dissertation / PhD Thesis | PreJuSER-996 |
2008
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/3222
Report No.: Juel-4280
Abstract: Corynebacterium glutamicum can utilize a variety of carbohydrates and organic acids. In contrast to other bacteria, C. glutamicum typically does not show diauxic growth on mixed carbon sources, but co-utilizes the present carbon sources. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during growth on substrate mixtures as compared to growth on glucose as sole carbon source. To cope with fast changing situations, especially with respect to nutrient availability, microorganisms have evolved a variety of different strategies to allow optimal growth. In this work the control of carbohyrate uptake and utilization in C. glutamicum was studied to extend the knowledge of complex gene regulation in this industrial important organism. Thereby, the DeoR-type transcriptional regulator SugR was identified as repressor of ptsG, ptsS and ptsF expression encoding the PTS permeases specific for glucose, sucrose and fructose uptake, regions. A partly conserved 8 bp SugR binding-motif was found in the corresponding promoter fragments. On gluconeogenic carbon sources, e.g. pyruvate or acetate, SugR represses ptsG transcription. During growth on PTS sugars, the identified effector, fructose-6-phosphate, prevents SugR from binding, thus ptsG is derepressed. Repression of SugR is maximal when fructose-6-phosphate concentrations are low, representing a mechanism which allows C. glutamicum to adapt glucose uptake to carbon source availability. Meanwhile, several transcriptional regulators of pts genes are known, among these are the two functionally equivalent regulators GntR1 and GntR2. GntR-type transcriptional regulators are involved in negative control of gluconate utilization genes in many bacteria and it was shown that gluconate itself interferes with their binding. A new and surprising aspect of this work was that GntR1 and GntR2 activate expression of ptsG and ptsS, too. This is the first example of pts gene control by gluconate. It is obvious that these transcriptional regulators are important players in a complex regulatory network, controlling uptake and metabolism of carbon sources in order to allow the most favourable combination of available substrates. In addition, the results indicate that regulation of the central carbon metabolism in C. glutamicum comprises control of the glyoxylate and TCA cycle genes by the global regulators RamA and RamB on the one side and control of glycolysis and pentose phosphate pathway genes by SugR on the other side. The SugR regulon to date comprises at least 17 genes in 14 transcription units. The genes encoding e.g. 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), NAD-dependent L-lactate dehydrogenase (ldhA) and transketolase (tkt) were identified as direct SugR targets and enzymatic activity measeurements revealed that SugR-mediated repression affects the activities of PfkA, LdhA, Pyk and Fba in vivo, whereas maximum SugR control is on LdhA activity. As SugR controls pts gene expression and the expression of genes involved in further consumption of these substrates in C. glutamicum, the gene encoding the initial step in sucrose metabolism, the sucrose-6-phosphate hydrolase (ScrB), was biochemically characterized and SugR-dependent control was analyzed. The results showed that SugR does not regulate scrB. Thus, control of ptsS and scrB with respect to the carbon source sucrose is clearly different. In C. glutamicum L-lactate is produced from pyruvate by LdhA. The L-lactate utilization operon cg3226-lldD, encoding the quinone-dependent L-lactate dehydrogenase, is essential for growth on L-lactate as sole carbon source. Control of L-lactate metabolism by the FadR-type regulator LldR on the one hand ensures that the L-lactate utilization operon, is expressed only when L-lactate is present. Whereas, SugR control of ldhA on the other hand ensures that ldhA expression is maximal when i.e. supply of carbohydrate growth substrates entering glycolysis is sufficient and therewith fructose-6-phosphate concentrations are high. Under oxygen deprivation conditions, overexpression of sugR reduced L-lactate formation by about 25% and sugR deletion improved L-lactate formation three fold. SugR negatively controls expression of pts genes and controls the glycolytic flux straight forward to pyruvate, which serves as precursor for L-lysine and L-lactate production in C. glutamicum, and also controls the flux through the pentose phosphate pathway leading to a better NADPH supply. Thus, this regulator is therefore a good candidate for improving L-lysine production with C. glutamicum. A sugR deletion in the strain DM1729 led indeed to an inceased lysine yield of about 20%, 50% and 70% on the PTS sugars glucose, fructose and sucrose, respectively.
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